RESUMO
A method named sequence-specific capture of oligonucleotide probes (SCOPE) was developed for quantification of microbial rRNA molecules in a multiplex manner. In this method, a molecular weight cutoff membrane (MWCOM) was used for the separation of fluorescence-labeled oligonucleotide probes hybridized with rRNA from free unhybridized probes. To demonstrate proof of concept, probes targeting bacteria or archaea at different taxonomic levels were prepared and were hybridized with rRNAs. The hybridization stringency was controlled by adjusting reaction temperature and urea concentration in the mixture. Then, the mixture was filtered through the MWCOM. The rRNA and hybridized probes collected on the MWCOM were recovered and quantified using a spectrophotometer and fluorospectrometer, respectively. The method showed high accuracy in detecting specific microbial rRNA in a defined nucleic acid mixture. Furthermore, the method was capable of simultaneous detection and quantification of multiple target rRNAs in a sample with sensitivity up to a single-base mismatch. The SCOPE method was tested and benchmarked against reverse transcription-quantitative PCR (RT-qPCR) for the quantification of Bacteria, Archaea, and some key methanogens in anaerobic sludge samples. It was observed that the SCOPE method produced more reliable and coherent results. Thus, the SCOPE method allows simple and rapid detection and quantification of target microbial rRNAs for environmental microbial population analysis without any need for enzymatic reactions. IMPORTANCE Microorganisms play integral roles in the Earth's ecosystem. Microbial populations and their activities significantly affect the global nutrient cycles. Quantification of key microorganisms provides important information that is required to understand their roles in the environment. Sequence-based analysis of microbial population is a powerful tool, but it provides information only on relative abundance of microorganisms. Hence, the development of a simpler and quick method for the quantification of microorganisms is necessary. To address the shortcomings of a variety of molecular methods reported so far, we developed a simple, rapid, accurate, and multiplexed microbial rRNA quantification method to evaluate the abundance of specific microbial populations in complex ecosystems. This method demonstrated high specificity, reproducibility, and applicability to such samples. The method is useful for quantitative detection of particular microbial members in the environment.
Assuntos
Archaea/genética , Bactérias/genética , Sondas de Oligonucleotídeos , RNA Ribossômico 16S/genética , Membranas Artificiais , Microbiota , Peso MolecularRESUMO
Oxidation of nitrite to nitrate is an important process in the global nitrogen cycle. Recent molecular biology-based studies have revealed that the widespread nitrite-oxidizing bacteria (NOB) belonging to the genus 'Candidatus Nitrotoga' may be highly important for the environment. However, the insufficient availability of pure Nitrotoga cultures has limited our understanding of their physiological and genomic characteristics. Here, we isolated the 'Ca. Nitrotoga' sp. strain AM1P, from a previously enriched Nitrotoga culture, using an improved isolation strategy. Although 'Ca. Nitrotoga' have been recognized as cold-adapted NOB, the strain AM1P had a slightly higher optimum growth temperature at 23°C. Strain AM1P showed a pH optimum of 8.3 and was not inhibited even at high nitrite concentrations (20 mM). We obtained the complete genome of the strain and compared the genome profile to five previously sequenced 'Ca. Nitrotoga' strains. Comparative genomics suggested that lactate dehydrogenase may be only encoded in the strain AM1P and closely related genomes. While the growth yield of AM1P did not change, we observed faster growth in the presence of lactate in comparison to purely chemolithoautotrophic growth. The characterization of the new strain AM1P sheds light on the physiological adaptation of this environmentally important, but understudied genus 'Ca. Nitrotoga'.
Assuntos
Gallionellaceae/fisiologia , Genoma Bacteriano , Crescimento Quimioautotrófico , L-Lactato Desidrogenase/genética , Ácido Láctico/metabolismoRESUMO
Two 600-bp DNA solutions (DNA600-G and DNA600-T) were developed as certified reference material, NMIJ CRM 6205-a, for the validation of DNA quantification methods. Both DNA600-G and DNA600-T are ideal as "spike-in control" because these materials have artificial nucleic acid sequences. The certified values were determined as the mass concentration of total DNA (whole DNA materials in sample solution regardless of sequence) at 25 °C by formic acid hydrolysis/liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) and inductively coupled plasma-mass spectrometry (ICP-MS) based on the amount of phosphorus. DNAs were synthesized, and plasmids including the synthesized DNAs were cloned into Escherichia coli DH5α. The amplified plasmids were digested with a restriction enzyme and highly purified. Then, the purified DNAs were diluted with water to approximately 1 ng/µL. By using the CRM-validated methods in fields where DNA quantification is required, the reliability of DNA quantification could be improved. Graphical abstract.
Assuntos
DNA/análise , Espectrometria de Massas/métodos , Sequência de Bases , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , DNA/genética , Formiatos/química , Hidrólise , Espectrometria de Massas/normas , Plasmídeos/análise , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Padrões de ReferênciaRESUMO
High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Metagenoma , Metagenômica , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/genética , Biologia Computacional , Microbiologia Ambiental , Metagenômica/métodos , Metagenômica/normas , Microbiota , Padrões de ReferênciaRESUMO
Candidate phylum TM6 is a major bacterial lineage recognized through culture-independent rRNA surveys to be low abundance members in a wide range of habitats; however, they are poorly characterized due to a lack of pure culture representatives. Two recent genomic studies of TM6 bacteria revealed small genomes and limited gene repertoire, consistent with known or inferred dependence on eukaryotic hosts for their metabolic needs. Here, we obtained additional near-complete genomes of TM6 populations from agricultural soil and upflow anaerobic sludge blanket reactor metagenomes which, together with the two publicly available TM6 genomes, represent seven distinct family level lineages in the TM6 phylum. Genome-based phylogenetic analysis confirms that TM6 is an independent phylum level lineage in the bacterial domain, possibly affiliated with the Patescibacteria superphylum. All seven genomes are small (1.0-1.5 Mb) and lack complete biosynthetic pathways for various essential cellular building blocks including amino acids, lipids, and nucleotides. These and other features identified in the TM6 genomes such as a degenerated cell envelope, ATP/ADP translocases for parasitizing host ATP pools, and protein motifs to facilitate eukaryotic host interactions indicate that parasitism is widespread in this phylum. Phylogenetic analysis of ATP/ADP translocase genes suggests that the ancestral TM6 lineage was also parasitic. We propose the name Dependentiae (phyl. nov.) to reflect dependence of TM6 bacteria on host organisms.
Assuntos
Bactérias Anaeróbias/genética , Filogenia , RNA Ribossômico 16S/genética , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Genoma Bacteriano , Interações Hospedeiro-Parasita/genética , Metagenoma/genética , Microbiologia do SoloRESUMO
Bioassay-guided separation of a lipophilic extract of the crinoid Alloeocomatella polycladia, inhibiting the activity of HCV NS3 helicase, yielded two groups of molecules: cholesterol sulfate and four new aromatic sulfates 1-4. The structures of the aromatics were elucidated by spectroscopic analysis in addition to theoretical studies. The aromatic sulfates 1-4 showed moderate inhibition against NS3 helicase with IC50 values of 71, 95, 7, and 5 µM, respectively.
Assuntos
Antivirais/farmacologia , Organismos Aquáticos/química , Equinodermos/química , RNA Helicases/antagonistas & inibidores , Sulfatos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Hepacivirus/efeitos dos fármacosRESUMO
A novel, strictly anaerobic, short rod-shaped bacterium, designated strain TBC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based organic wastewater. Cells of this strain were 2-4 µm long and 0.4-0.6 µm wide. They were non-motile and Gram-stain-negative. The optimum growth temperature was 30-37 °C, with a range of 20-40 °C. The optimum pH for growth was around pH 7.0, while growth occurred in a range of pH 6.5-9.0. Strain TBC1T grew chemo-organotrophically on a narrow range of carbohydrates under anaerobic conditions. Yeast extract was required for its growth. The major fermentative end products from glucose, supplemented with yeast extract, were acetate, malate, propionate, formate and hydrogen. Doubling time under optimal growth conditions was estimated to be 1 day. The DNA G+C content of strain TBC1T was 49.2 mol% as determined by HPLC. Major cellular fatty acids were C16 : 0, C18 : 0, C16 : 1ω9c and C18 : 1ω9c. Based on its 16S rRNA gene sequence, strain TBC1T was shown to represent a distinct lineage at the family level in the phylum Bacteroidetes. Among previously described species of this phylum, Mucilaginibacter boryungensis BDR-9T (Sphingobacteriaceae) displayed the highest sequence similarity (85.9 %) with strain TBC1T. Phylogenomic analyses using 38-83 single copy marker genes also supported the novelty of strain TBC1T at the family level. Based on its characteristics, strain TBC1T (=JCM 30898T=DSM 100618T) is considered to be the type strain of a novel species of a new genus, Lentimicrobium saccharophilum gen. nov., sp. nov. A new family, Lentimicrobiaceae fam. nov., is also proposed encompassing the strain and related environmental 16S rRNA gene clone sequences.
Assuntos
Bacteroidetes/classificação , Filogenia , Esgotos/microbiologia , Águas Residuárias/microbiologia , Técnicas de Tipagem Bacteriana , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Fermentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
A novel obligately anaerobic bacterium, designated strain TC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based wastewater. Cells had a multicellular filamentous morphology, stained Gram-negative and were non-motile. The filaments were flexible, generally >100 µm long and 0.3-0.4 µm wide. Growth of the isolate was observed at 25-43 °C (optimum 37 °C) and pH 6.0-8.5 (optimum pH 7.0). Strain TC1T grew chemo-organotrophically on a range of carbohydrates under anaerobic conditions. Yeast extract was required for growth. The major fermentative end products of glucose, supplemented with yeast extract, were acetate, lactate, succinate, propionate, formate and hydrogen. Co-cultivation with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T enhanced growth of the isolate. The DNA G+C content was determined experimentally to be 42.1âmol%. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C17 : 0 3-OH. Based on 16S rRNA gene sequence analysis, strain TC1T belonged to the class Anaerolineae in the phylum Chloroflexi, in which Ornatilinea apprima P3M-1T was its closest phylogenetic relative (88.3 % nucleotide identity). Phylogenomic analyses using 38 and 83 single-copy marker genes also supported the novelty of strain TC1T at least at the genus level. Based on phylogenetic, genomic and phenotypic characteristics, we propose that strain TC1T represents a novel species of a new genus, for which we suggest the name Flexilinea flocculi gen. nov., sp. nov. The type strain of Flexilinea flocculi is strain TC1T ( = JCM 30897T = CGMCC 1.5202T).
RESUMO
How aromatic compounds are degraded in various anaerobic ecosystems (e.g. groundwater, sediments, soils and wastewater) is currently poorly understood. Under methanogenic conditions (i.e. groundwater and wastewater treatment), syntrophic metabolizers are known to play an important role. This study explored the draft genome of Syntrophorhabdus aromaticivorans strain UI and identified the first syntrophic phenol-degrading phenylphosphate synthase (PpsAB) and phenylphosphate carboxylase (PpcABCD) and syntrophic terephthalate-degrading decarboxylase complexes. The strain UI genome also encodes benzoate degradation through hydration of the dienoyl-coenzyme A intermediate as observed in Geobacter metallireducens and Syntrophus aciditrophicus. Strain UI possesses electron transfer flavoproteins, hydrogenases and formate dehydrogenases essential for syntrophic metabolism. However, the biochemical mechanisms for electron transport between these H2 /formate-generating proteins and syntrophic substrate degradation remain unknown for many syntrophic metabolizers, including strain UI. Analysis of the strain UI genome revealed that heterodisulfide reductases (HdrABC), which are poorly understood electron transfer genes, may contribute to syntrophic H2 and formate generation. The genome analysis further identified a putative ion-translocating ferredoxin : NADH oxidoreductase (IfoAB) that may interact with HdrABC and dissimilatory sulfite reductase gamma subunit (DsrC) to perform novel electron transfer mechanisms associated with syntrophic metabolism.
Assuntos
Deltaproteobacteria/genética , Deltaproteobacteria/metabolismo , Transporte de Elétrons/fisiologia , Anaerobiose/fisiologia , Carbono-Carbono Liases/metabolismo , Carboxiliases/metabolismo , Deltaproteobacteria/classificação , Elétrons , Ferredoxinas/metabolismo , Formiato Desidrogenases/metabolismo , Formiatos/metabolismo , Genoma Bacteriano/genética , Hidrogenase/metabolismo , Oxirredutases/metabolismo , Fenol/metabolismoRESUMO
RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration-response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT-PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT-PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.
Assuntos
Modelos Químicos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Sondas RNA/química , Padrões de ReferênciaRESUMO
Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure-activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.
Assuntos
Antracenos/farmacologia , Antraquinonas/farmacologia , Antivirais/farmacologia , Hepacivirus/enzimologia , Perileno/análogos & derivados , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Antracenos/química , Antraquinonas/química , Antivirais/química , Linhagem Celular , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Perileno/química , Perileno/farmacologia , RNA Helicases/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
A mesophilic, obligately anaerobic, lactate-, alcohol-, carbohydrate- and amino-acid- degrading bacterium, designated strain 7WAY-8-7(T), was isolated from an upflow anaerobic sludge blanket reactor treating high-strength organic wastewater from isomerized sugar production processes. Cells of strain 7WAY-8-7(T) were motile, curved rods (0.7-1.0×5.0-8.0 µm). Spore formation was not observed. The strain grew optimally at 37 °C (range for growth was 25-40 °C) and pH 7.0 (pH 6.0-7.5), and could grow fermentatively on yeast extract, glucose, ribose, xylose, malate, tryptone, pyruvate, fumarate, Casamino acids, serine and cysteine. The main end-products of glucose fermentation were acetate and hydrogen. In co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864(T), strain 7WAY-8-7(T) could utilize lactate, glycerol, ethanol, 1-propanol, 1-butanol, L-glutamate, alanine, leucine, isoleucine, valine, histidine, asparagine, glutamine, arginine, lysine, threonine, 2-oxoglutarate, aspartate and methionine. A Stickland reaction was not observed with some pairs of amino acids. Yeast extract was required for growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite and Fe (III) were not used as terminal electron acceptors. The G+C content of the genomic DNA was 61.4 mol%. 16S rRNA gene sequence analysis revealed that the isolate belongs to the uncultured environmental clone clade (called 'PD-UASB-13' in the Greengenes database) in the bacterial phylum Synergistetes, showing less than 90% sequence similarity with closely related described species such as Aminivibrio pyruvatiphilus and Aminobacterium colombiense (89.7% and 88.7%, respectively). The major cellular fatty acids were iso-C(13â:â0), iso-C(15â:â0), anteiso-C(15â:â0), C(18â:â1), C(19â:â1), C(20â:â1) and C(21â:â1). A novel genus and species, Lactivibrio alcoholicus gen. nov., sp. nov. is proposed to accommodate strain 7WAY-8-7(T) (â=âJCM 17151(T)â=âDSM 24196(T)â=âCGMCC 1.5159(T)).
Assuntos
Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/classificação , Filogenia , Esgotos/microbiologia , Álcoois/metabolismo , Técnicas de Tipagem Bacteriana , Composição de Bases , Metabolismo dos Carboidratos , DNA Bacteriano/genética , Ácidos Graxos/química , Fermentação , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/genética , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/isolamento & purificação , Ácido Láctico/metabolismo , Dados de Sequência Molecular , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Eliminação de Resíduos LíquidosRESUMO
Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC50 = 1.7 ± 0.2 µM with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3-RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.
Assuntos
Antivirais/farmacologia , Ésteres do Colesterol/farmacologia , Hepacivirus/efeitos dos fármacos , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Antivirais/isolamento & purificação , Organismos Aquáticos/química , Ésteres do Colesterol/isolamento & purificação , Relação Dose-Resposta a Droga , Descoberta de Drogas , Hepacivirus/enzimologia , Estrutura Molecular , Ligação Proteica , Serina Proteases/metabolismoRESUMO
Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.
Assuntos
Hepacivirus/enzimologia , Naftalenos/química , Naftalenos/farmacologia , Poríferos/metabolismo , Inibidores de Serina Proteinase/farmacologia , Sesterterpenos/química , Sesterterpenos/farmacologia , Ésteres do Ácido Sulfúrico/química , Ésteres do Ácido Sulfúrico/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Elétrons , Naftalenos/isolamento & purificação , RNA Viral/metabolismo , Serina Proteases/química , Sesterterpenos/isolamento & purificação , Ésteres do Ácido Sulfúrico/isolamento & purificaçãoRESUMO
The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3) is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE) 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1) on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1) against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Éteres Difenil Halogenados/farmacologia , Poríferos/química , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenosina Trifosfatases/química , Animais , Antivirais/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Éteres Difenil Halogenados/isolamento & purificação , Hepacivirus/química , Hepacivirus/enzimologia , Humanos , RNA Helicases/química , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.
Assuntos
Adenovírus Humanos/isolamento & purificação , Água Doce/microbiologia , Microfluídica/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Microbiologia da Água , Intestinos/virologia , RiosRESUMO
Mycobacteroides (Mycobacterium) abscessus, which causes a variety of infectious diseases in humans, is becoming detected more frequently in clinical specimens as cases are spreading worldwide. Taxonomically, M. abscessus is composed of three subspecies of M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, with different susceptibilities to macrolides. In order to identify rapidly these three subspecies, we determined useful biomarker proteins, including ribosomal protein L29, L30, and hemophore-related protein, for distinguishing the subspecies of M. abscessus using the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiles. Thirty-three clinical strains of M. abscessus were correctly identified at the subspecies-level by the three biomarker protein peaks. This study ultimately demonstrates the potential of routine MALDI-MS-based laboratory methods for early identification and treatment for M. abscessus infections.
Assuntos
Proteínas de Bactérias , Mycobacterium abscessus , Proteínas Ribossômicas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/análise , Mycobacterium abscessus/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Biomarcadores/análise , Biomarcadores/metabolismoRESUMO
Wickerhamiella is a genus of budding yeast that is mainly isolated from environmental samples, and 40 species have been detected. The yeast isolated from human clinical samples usually only contain three species: W. infanticola, W. pararugosa and W. sorbophila. In this study, we isolated W. tropicalis from a blood sample of a six-year-old female with a history of B-cell precursor lymphoblastic leukemia in Japan in 2022. Though the strain was morphologically identified as Candida species by routine microbiological examinations, it was subsequently identified as W. tropicalis by sequencing the internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The isolate had amino acid substitutions in ERG11 and FKS1 associated with azole and echinocandin resistance, respectively, in Candida species and showed intermediate-resistant to fluconazole and micafungin. The patient was successfully treated with micafungin. Furthermore, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detected three novel peaks that are specific for W. tropicalis, indicating that MALDI-MS analysis is useful for rapid detection of Wickerhamiella species in routine microbiological examinations.
Assuntos
Antifúngicos , Saccharomycetales , Feminino , Humanos , Criança , Antifúngicos/farmacologia , Hemocultura , Micafungina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Testes de Sensibilidade Microbiana , CandidaRESUMO
A mesophilic, obligately anaerobic, carbohydrate-fermenting bacterium, designated 8KG-4(T), was isolated from an upflow anaerobic sludge blanket reactor treating high-strength organic wastewater from salted vegetable production processes. Cells of strain 8KG-4(T) were non-motile, spherical and 0.7-1.5 µm in diameter (mean, 1.0 µm). Spore formation was not observed under any culture conditions tested. The strain grew optimally at 37 °C (range for growth 25-40 °C) and pH 7.0 (range, pH 6.5-7.5), and could grow fermentatively on glucose, ribose, xylose, galactose and sucrose. The main end products of glucose fermentation were acetate, ethanol and hydrogen. Organic acids, alcohols and amino acids were not utilized for growth. Yeast extract was not required for growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite and Fe(III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of the genomic DNA was 61.1 mol%. 16S rRNA gene sequence analysis revealed that the isolate represented a previously uncultured lineage at the subphylum level within the phylum Lentisphaerae known as 'WWE2 subgroup I'. The major cellular fatty acids were anteiso-C(15â:â0), iso-C(16â:â0), C(16â:â0) and anteiso-C(17â:â0). Respiratory quinones were not detected. The most abundant polar lipid of strain 8KG-4(T) was phosphatidylethanolamine. A novel genus and species, Oligosphaera ethanolica gen. nov., sp. nov., is proposed to accommodate strain 8KG-4(T) (â=âJCM 17152(T)â=âDSM 24202(T) â=âCGMCC 1.5160(T)). In addition, we formally propose Oligosphaeria classis nov. and the subordinate taxa Oligosphaerales order nov. and Oligosphaeraceae fam. nov.
Assuntos
Bactérias Anaeróbias/classificação , Metabolismo dos Carboidratos , Filogenia , Esgotos/microbiologia , Bactérias Anaeróbias/genética , Bactérias Anaeróbias/isolamento & purificação , Bactérias Anaeróbias/metabolismo , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/análise , Fermentação , Dados de Sequência Molecular , Fosfatidiletanolaminas/análise , Quinonas/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
We report a complete genome sequence of Anaerostipes hadrus JCM 17467T. The genome consists of a circular chromosome of 2,804,089 bp, with a G+C content of 37.3%. The genome was predicted to contain 21 rRNA genes, 65 tRNA genes, and 2,675 protein-coding sequences.